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Mycoplasma arthritidis mitogen. V engaged in mice rats and humans and requirement of HLA-DR╨Ю┬▒ for presentation

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MYCOPLASMA ARTHRITIDIS MITOGEN
Vp Engaged in Mice, Rats, and Humans, and Requirement of
HLA-DRa for Presentation
ROBERTO BACCALA, LAWRENCE R. SMITH, MIKAEL VESTBERG, PER A. PETERSON,
BARRY C. COLE, and ARGYRIOS N. THEOFILOPOULOS
Objective. Mycoplasma arthritidis mitogen
(MAM) is mitogenic for mouse, rat, and human T cells,
and behaves as a superantigen in mice through its
capacity to bind to the (Y chain of I-E molecules and
engage entire sets of T cells expressing specific Vp.Here,
we have attempted to fully characterize the V,-engaging
activities of MAM in mice, and define similar activities
in rats and humans.
Methods. Multiprobe RNase-protection assays
and mice transgenic for human DRcu, DRP, and DRaP
were utilized for this purpose.
Results. MAM-reactive V’ in the mouse included
not only the previously reported V&, Vp8.1, Vp8.2, and
V9.3, but also V,5.1. In the rat, engagement of Vs.1,
V&, V9.1, and V’8.2, but not Vp8.3, was documented,
, included primarily
whereas in humans, the engaged V
V,19.1 (alternatively termed V’17.1) and, to a lesser
~..
Publication No. 7007-1MM from the Department of Immunology, the Research Institute of Scripps Clinic, La Jolla, CA.
From the Department of Immunology, the Research Inslitute of Scripps Clinic, La Jolla, California. and the University (of
Utah School of Medicine, Salt Lake City.
Supported in part by NIH grants AR-31203, AR-39555.
CA-52539, AG-09430, AI-26610, AI-12103, and AR-02255.
Roberto Baccala, PhD: Department of Immunology, Research Institute of Scripps Clinic; Lawrence R. Smith, PhD: Department of Immunology, Research Institute of Scripps Clinic
(current address: Immune Response Corp., Carlsbad, CA); Mikael
Vestberg, PhD: Department of Immunology, Research Institute of
Scripps Clinic; Per A. Peterson, MD, PhD: Department of Immunology, Research Institute of Scripps Clinic; Barry C. Cole, PhD:
University of Utah School of Medicine: Argyrios N . Theofilopoulos,
MD: Department of Immunology, Research Institute of Scripps
Clinic.
Address reprint requests to Argyrios N . Theofilopoulos,
MD, Immunology Department, Scripps Research Institute, 10666
North Torrey Pines RoadIIMM3. La Jolla. CA 92037.
Submitted for publication September 5 , 1991; accepted in
revised form November 5 . 1991.
Arthritis and Rheumatism, Vol. 35, No. 4 (April 1992)
extent, Vp3.1, VDl1.l, V32.1, and V,13.1. In DR transgenic Ea-EP- mice, presentation of MAM and engagement of specific Vs was effected by DRa.
Conclusions. Homologous ,
V are engaged by
MAM in mice, rats, and humans, presumably through a
binding site similar to that proposed previously for other
superantigens. MAM presentation primarily via the
nonpolymorphic DRa makes it unlikely that there is
involvement of such a superantigen in the pathogenesis
of autoimmune diseases known to be associated with
certain DR haplotypes. The possibility cannot be excluded, however, that superantigen-activated T cells
may lead to disease by cross-reactions with self-antigens
presented by particular DR haplotypes.
Mycoplasmds, the smallest self-replicating prokaryotes, are parasites of humans, animals, and plants
(1). Although some mycoplasmas are part of the
normal flora of mammals, most species cause a variety
of pathologic manifestations, of which arthritis is
frequent (for review, see ref. 2). Consequently, a
possible link of mycoplasma infections to human rheumatoid arthritis (RA) has been sought, although the
significance of the reported findings (3-6) remains a
subject of controversy (7).
Among the arthritogenic mycoplasmas, Mycoplasma arthritidis is one of the best characterized
(2,8), yet the precise mechanisms by which this microorganism avoids host defenses and induces arthritis in
rodents remains to be defined. It has been known for
some time, however, that a soluble factor produced by
M arthritidis can act as a potent mitogen for mouse,
rat, and human T cells (9,lO). This factor, the molecular characteristics of which have not been fully
defined, has been termed M urthritidis mitogen
Vp AND MHC ENGAGED BY MAM
(MAM) and recently classified as a superantigen
(11,12). The term superantigen was coined to denote
molecules that need not be processed (13-16), bind
outside the classical peptide-binding groove of class I1
major histocompatibility complex (MHC) molecules
(17,18) and stimulate almost entire sets of T cells
expressing specific T cell receptor V, gene products
(19). A variety of bacterial and viral products have
been documented to act as superantigens (for review,
see refs. 20 and 21). Superantigen-induced T cell
stimulations could produce direct toxic consequences,
compromise the host immune response to the invading
microbe, or even cause bystander effects such as
tissue damage by expanding self-reactive T cell clones
(for review, see refs. 20, 22, and 23). Since RA is
associated with significant synovial T cell infiltration,
the hypothesis has been made that superantigen-like
molecules might be the inducing agents, either directly
through local sequestration in the joint or indirectly by
activation of extraarticular T cells that are reactive
with synovial antigens. Therefore, definition of the
V,-selecting properties of bacterial and/or viral molecules and characterization of the clonal diversity of the
T cells that infiltrate the inflamed joints have been
central to our current efforts to identify the causes and
mechanisms of induction of RA.
Previous studies of mice, for which a limited set
of monoclonal antibodies (MAb) was used, showed
that MAM selectively activated T cells expressing V&,
V,S.l, V,8.2, and V,8.3 (11,12). In the present work,
using a large number of V,-specific riboprobes in an
RNase-protection assay, we identified an additional
MAM-reactive V, in mice and characterized for the
first time the V, engaged by MAM in rats and humans.
Furthermore, using transgenic mice, we documented
that MAM presentation by human class I1 MHC
molecules is primarily mediated through the nonpolymorphic DRa chain.
MATERIALS AND METHODS
Lymphocyte suspensions. Mice (CBA/CaJ, I-Ek,
Mlsb; BlO.M, I-E-, Mlsb) were either bred in the Animal
Resource Facility of the University of Utah or in the Scripps
Clinic vivarium. BI0.M mice transgenic for human class I1
MHC DRA*0101 a chain (DRaB10.M) or DRB*0301 /3 chain
(DRpB10.M) or both (DRapB10.M) were generated as described elsewhere (24). Rats (Lewis, RT1’) were obtained
from the Scripps Breeding Colony. Spleen lymphocyte suspensions were prepared in RPMI 1640 medium (Whittaker,
Walkersville, MD) as described elsewhere (25).
Human peripheral blood lymphocytes (PBL) were
435
prepared from 2 MHC-typed normal donors (PBL#I : A1 I ;
B27/55;C1/2;DR14/15,DRw52;DQ5/6
and PBL#2: A24;B7/
47;C3;DRl1115,DRw52;DQ6/7)
by Ficoll-Hypaque discontinuous gradient centrifugation.
Lymphocyte stimulation. Mouse, rat, and human lymphocytes (2.5 x lo6 per ml) were cultured in RPMI 1640
containing 10% fetal calf serum, 5 x lo-’ mM 2-mercaptoethanol, and 2 mM L-glutamine. Partially purified MAM was
prepared according to a modification of the procedure previously described (26) and involved 2 additional ionexchange chromatographic steps (Atkin CL, Wells DJ, Cole
BC: unpublished). MAM was used at a final dilution of
1:25,OOO, which had been determined to be optimal for T cell
proliferation. After 3 days at 37°C in 6% CO,, cultures were
supplemented with 20 units/ml of recombinant human interleukin-2 (IL-2) (Genzyme, Boston, MA) and incubated for an
additional 24 hours. The cells were then centrifuged, and the
cell pellets were stored at -80°C until used.
Multiprobe RNase protection assays. Generation of
mouse (25), rat (Smith et al: unpublished observations), and
human (27) 5-specific riboprobes and of the corresponding
multiprobe RNase-protection assays have been described.
Briefly, the assay was performed as follows: 5-10 pg of
lyophilized total lymphocyte RNA (28) was dissolved in S pl
of hybridization buffer (80% formamide, 0.4M NaCI, 1 mM
EDTA, 40 mM PIPES, pH 6.7) containing 1 of the radiolabeled probe sets (see Results). Following hybridization at
56°C for 12-16 hours, unhybridized probes and target RNA
were digested using 50 pl of digestion buffer (10 mM Tris, pH
7.95 mM EDTN0.3M NaCI) containing 5 &ml RNase A
and 10 unitslml RNase TI. After 1 hour at 37”C, digested
samples containing “protected” probe-RNA duplexes were
phenol-chloroform extracted, ethanol precipitated, dissolved in sample buffer, and electrophoresed in standard 6%
polyacrylamide sequencing gels. Autoradiography of the
dried gel was done on Kodak XRP film at -7O”C, with
intensifying screens, for 10-20 hours.
Quantitation of Va transcript levels was performed
using an Ambis radioanalytic imaging apparatus (Ambis
Systems, San Diego, CA). The net counts per minute at a
given band corresponding to a specific protected Vs probe
was calculated by the formula [(cpm of Vp-specific band) (cpm of background around the band)]/(number of uridine
residues in the specific Vp probe). This value was then
expressed as the percentage of total Vp-transcripts (mouse
and rat assays) or Cp-transcripts (human assay).
RESULTS
Mouse, rat, and human ,
V gene products selected
by MAM. Using a limited set of mouse V, (mV,)specific monoclonal antibodies, it has been established
that T cells expressing mVp6, mV,8.1, mV,8.2, and
mV,8.3 are strongly enriched in murine splenocyte
cultures incubated with MAM (1 1,12). To determine
whether additional mV, gene products are selected by
this mitogen, we analyzed MAM-stimulated splenocyte cultures from I-E+ CBNCaJ mice by a multi-
BACCALA ET AL
436
probe RNase-protection assay encompassing 17 mVb
genes. In these studies, interpretations related to Cb
selection are based on overall V, expression profiles.
Nonengaged V, are represented by transcript levels
that are significantly reduced compared with those in
unstimulated cells, whereas engaged V, are represented by transcript levels similar to or higher than
those in unstimulated cells. As shown in Table 1, we
confirmed the engagement of V&, V,S.l, Vp8.2, and
V$.3 and noted engagement of mV,5.1 as well. Based
on relative changes in transcript levels from nonstimulated to stimulated cells, it appears that mVp6,
mVp8.2, and mV,5.1 are the strongest engaged by
MAM, followed by rnV9.l and mVp8.3.
To determine whether rats, which have an
overall V, genomic composition almost identical to
that of mice (29), respond to MAM in a similar
manner, we assessed the responses of spleen cells
from a strain of rats known to be a high responder to
this mitogen (Lewis rats) (10,30). As indicated in
Figure 1, transcript levels for rat V, (rV& 5.1, 6, 8.1,
and 8.2 were strongly enriched in these cultures but
rVp8.3, in contrast to mV,8.3, was not engaged.
MAM-stimulated human PBL were also characterized, and the results were virtually the same in
the 2 normal subjects studied (Figure 2). High MAM
reactivity was observed for hVf19.1 (nomenclature
according to ref. 31), also termed hV,17.1 (nomenclature according to ref. 32), which exhibits the highest
Table 1. Mouse \ (my) gene products engaged by Mycoplu.vmu
urtCitidis mitogen (MAM)*
C B N C a l (I-E+)
Control
MAM
0.38
I .64
0.50
0.62
4.26*
0.28
8.20t
0.25
32. IOt
30.60t
16.87t
0.38
0.14
0.53
I .oo
1.45
0.81
1
2.70
9
1 I .75
3. I
4
5. I
5.2
6
7
8.1
8.2
8.3
10
I1
12
13
14
15
1.87
2.23
2.01
0.29
I .65
0.49
21.13
16.24
17.35
2.90
0.25
0.99
3.20
9.03
5.91
-
* Values are the percentages of total rn\ transcripts.
t Values for m\-expressing clones selectively expanded by MAM.
A.
J
&
*B
Es
c 3
3.3 1%I
er
% of total VO
B.
o
m
o- v
-l 8
Nl
n
Wo
W
m
1514 I
81
r
8.2
Q€
I
Figure 1. Rat V, (r\) gene products engaged by Mycoplusmu urrhriridis mitogen (MAM). Total RNA extracted from nonstirnulated
(non st.) (open bars) or MAM-stimulated (solid bars) splenic rat
lymphocytes (Lewis strain) was analyzed by an RNase-protection
assay using 18 rat \-specific riboprobes, which based on size, were
organized into 2 probe sets. A, Autoradiographic profiles. B, Quantitative analysis performed as described in Materials and Methods.
Results are expressed as a percentage of total \ transcripts.
homology (69%) with mouse and rat Vp6. In addition,
lower but significant engagement was detected for
hVp3.1, hVpll.I, hV,l2.1, and hV,13.1, i.e., the hV,
genes most homologous (5464%) to the mouse and rat
V8' gene products. However, hV,l. 1 the most homologous hV' gene to mouse and rat V,5. I (58% homology), was not engaged.
V, binding site for MAM. Among the members
of mouse and rat $8 subfamilies, rVp8.3 is the only one
that is not engaged by MAM. To attempt to identify
the V, binding site for MAM, we compared the amino
acid sequence of the non-MAM-binding rVp8.3 with
that of the MAM-binding mouse and rat Vp8 gene
products. As shown in Figure 3, nine amino acids are
unique to rVp8.3. Of these, 5 are located in the first and
second hypervariable regions (HVR- 1 and HVR-2),
i.e., in regions predicted to interact with conventional
antigenic peptide-MHC complexes (33,34), whereas
the remaining 4, i.e., residues 17, 21, 22, and 71
(numbering according to ref. 3 3 , are located in regions
previously suggested to be critical for binding of
certain exogenous (staphylococcal enterotoxins C2
[SEC2] [see ref. 361) and endogenous (Mlsl" [see refs.
37 and 381) superantigens.
437
Vp AND MHC ENGAGED BY MAM
c
A.
G
ti
n3
w
B.
o
o
Of
d
co
G
8
. . . . . . . . . . . . . . . . . . . . .
17.18A18.119.111:15.1-
6.4
13.2
3.1
1.1
7.1
*
I
4.14
11.1-
12.1
111
14.11
5.1
17.1
iai
3 5
19.1
Figure 2. Human % (hVP) gene products engaged by MAM. Total
RNA extracted from nonstimulated (open bars) or MAM-stimulated
(solid bars) peripheral blood lymphocytes from a normal individual
(PBL#I) was analyzed by an RNase protection assay using 22
human 5-specific riboprobes organized into 3 probe sets. A, Autoradiographic profiles. B, Quantitative analysis. Results (mean and
SD of 2 assays) are expressed as a percentage of total C, trdnscripts.
Human %nomenclature is according to Wilson et al(31). See Figure 1
for other definitions.
The presence of N-linked carbohydrates on the
lateral surface of the V, has been found to affect the
binding of mV,8.2 to the self-superantigen Mlsl" (38).
The tripeptide sequence N-X-(S/T) (X being any amino
acid) represents the consensus sequence for N-linked
glycosylation. As indicated in Figure 3, all known
members of the mouse and rat V$ families have one
potential glycosylation site, except for mV,8.2, which
has two. The non-MAM-reactive rVB8.3 has a potential glycosylation site (residue 18) that is identical to
that of the MAM-reactive 1nV~8.3,and therefore, it
cannot be a determining factor in MAM reactivity.
MAM binds to the a-chain of HLA-DR. In mice,
presentation of MAM to T cells is associated with the
expression of I-E molecules on accessory cells (9), and
is restricted by determinants associated with the I-E
a-chain (39). In human lymphocytes, there is evidence
that MAM associates with the Q chain of HLA-DR
(the human equivalent of mouse Ea) since MAb to the
conserved HLA-DR a chain inhibits proliferative responses to MAM (23). In addition, studies with transgenic mice have shown that DRcv in association with
endogenous EP is able to present mouse mammary
tumor virus (MMTV) integrant-encoded self-superantigens and induce the corresponding mV, clonal deletions (24). We, therefore, analyzed responsiveness to
MAM by spleen lymphocytes from I-E- (Ea-EP-)
BIO.M mice rendered transgenic for either DRa or
DRP, or both, to identify the DR chain responsible for
MAM binding and presentation.
Cell surface expression of DRa and/or DRP
transgenes was determined by immunofluorometric
and immunoprecipitation analysis (not shown). Double transgenic splenocytes expressed normal levels of
cell surface DR molecules. Similarly, in DRatransgenic splenocytes, D R a likely paired with the
endogenous A@ chain, forming surface-displayed interspecies D R e A P f hybrid molecules, albeit to a
lesser extent than in the homologous DRcr-DRP pairing. In contrast, in DRPtransgenic splenocytes, DRP
was detected only intracellularly , indicating either that
DRP cannot pair with A a or, less likely, that conformational changes, which are induced in the DRP chain
upon binding to A a , affected the recognition of DRP
on cell surfaces by the anti-DRP monoclonal antibody.
Splenocytes from control and transgenic mice
were cultured in the presence of MAM, and Vp levels
were analyzed by the RNase protection assay (Table
2). Unstimulated splenocytes of D R a and DRQP transgenic mice showed decreased levels of expression for
Vpl 1 and b 1 2 compared with nontransgenic I-E- mice
or to DRP-transgenic mice. These changes are pre-
definitions.
BACCALA ET AL
438
Table 2.
Presentation of Mycoiplusrnrr urthritidis mitogen (MAM) is restricted to the a chain of HLA-DR molecules*
tr-
my
1
2
3.1
4
5.1
5.2
6
7
8.1
8.2
8.3
10
I1
12
13
14
I5
DRptr
DRa-tr
-
DRaptr
Nonstimulated
MAMstimulated
Nonstimulated
MAMstimulzited
Nonstimulated
MAMstimulated
Nonstimulated
MAMstimulated
0.79
7.88
3.68
3.08
3.85
2.09
1.27
2.39
17. I5
21.32
14.03
1.38
4.14
2.18
5.92
6.44
2.40
0.25
9.88
2.30
3.80
3.35
0.25
1.93
1.69
16.09
27.75
11.13
2.43
4.55
I .69
6. I7
4.34
2.40
I .59
8.04
3.64
3.41
4.50
1.63
1.35
2.55
18.61
21.72
13.02
0.75
3.33
2.15
5.78
4.68
2.66
0.68
10.69
2.54
4.02
3.49
0.61
2.30
1.87
21.26
21.46
11.24
1.24
4.36
1.26
5.96
4.49
2.53
0.60
6.28
2.89
4.80
3.62
1.20
1.10
0.82
12.02
36.67
14.56
I .23
0.32
0.82
4.00
7.88
1.18
0.14
I .40
0.46
0.51
3.30t
0.01
3.48t
0.61
33.40t
42.05t
I I .94t
0.23
0. I5
0.27
0.84
1.14
0.05
0.59
7.03
2.83
3.35
3.93
2.23
1.96
1.32
12.86
34.20
13.75
1.80
0.63
0.75
2.82
8.03
I .93
0.92
I .23
0.01
0.67
3.14"
0.81
4.41t
0.45
28.56t
39.99t
15.74t
0.13
0.70
0.38
0.77
1.51
0.58
* Values are the percentages of total mouse % (mVd transcripts in nonstimulated and MAM-stimulated splenic lymphocytes from control (tr-)
and transgenic (tr) B1O.M mice expressing human DRP or DRa. or both.
t Selective engagement of splenic T cells expressing m55. I , mb6, mV$. I, mx8.2. and mV3.3 was noted in MAM-stimulated DRa and DRaP
transgenic mice.
sumably the result of binding and recognition of
MMTV integrant-encoded endogenous superantigens
(Lawrence SK, Peterson PA, Theofilopoulos AN:
unpublished observations). V, transcript levels in
MAM-stimulated splenocytes from DRP-transgenic
mice (DR surface negative) were similar to those in
unstimulated cultures. In contrast, MAM-stimulated T
cells from DRa or DRaP transgenic mice, expressing
surface DRa-AP or DR+DRP dimers, showed selective
representation of specific mV,-expressing clones. Interestingly, MAM engaged the same mV, gene products
regardless of whether it was presented in the context of
E-EP (Table I), DRcl-AP, or DRa-DRP dimers.
DISCUSSION
In the present study, we have further documented the superantigen-like activities of MAM and
characterized the V, selected by this molecule on T
cells from 3 species known to respond to this mitogen.
Since our panels of V, riboprobes encompass >95% of
the total expressed mouse and rat V, genes (25,29), it
can be stated that the identified V, constitute the
complete set of T cell clones engaged by this mitogen
in these 2 species. In contrast, since our panel of 22
human V, riboprobes encompass -45% of the known
expressed human V, genes (refs. 27, 40, and 41, and
Plaza AP, Kono DH, Theofilopoulos AN: unpublished
observations), the identified MAM-engaged human V,
may not constitute the full set of T cell clones involved
in the response to this mitogen. The possibility that
additional human V, are engaged by MAM is suggested
by the fact that the sum of MAM-induced V, transcripts accounted for only 30% to 45% of the total C,
transcripts.
Our results indicate that homologous V, are
engaged by MAM in mice, rats, and humans. Thus,
hV,19.1 is the most homologous to mouse and rat V'6,
while hV,3.I, hV,ll.l, hV,12.1, and hVJ3.1 are most
homologous to mouse and rat V,S. 1, Vp8.2, and Vp8.3
(3 1). Nevertheless, these V, exhibit a considerable
level of amino acid sequence divergence. For example, hVp19. 1, the strongest MAM-engaged hV, identified, displays 31% amino acid differences from the
MAM-reactive mouse and rat V,6. This finding, together with the diverse V, engaged, indicate that MAM
and other superantigens (38) are relatively tolerant to
extensive structural variations in the TCR V, molecules to which they bind.
Sequence comparisons and mutagenesis studies
have suggested that the TCR V' binding sites for 1
exogenous superantigen, reactive with hVp13.2 (36),
and for 2 endogenous superantigens, one reactive with
Vp8.2b (37,38) and the other with V,17a2 (42), are
located outside the V, hypervariable regions predicted
to bind conventional peptide-MHC complexes (34).
Vp AND MHC ENGAGED BY MAM
Based on amino acid differences between the MAM
nonreactive rVp8.3 and the MAM-reactive members of
the mouse and rat VpS subfamily, it appears that MAM
binds to similar sites as those proposed for other
superantigens, i.e., around residues 17-24 and 70-80
(numbering according to ref. 35) or residues 15-22 and
67-77 (numbering according to ref. 43). However,
since the nonreactive rVp8.3 exhibits unique residues
in other positions, some within HVR-1 and HVR-2, it
is possible that the combining site for MAM extends
over a larger area, partially overlapping the TCR’s
combining site for conventional peptide-MHC complexes. This latter possibility is further suggested by
recent mutagenesis studies by Pullen et a1 (38),
wherein 1 amino acid replacement at HVR-1 (position
26; numbering according to ref. 35) abolished binding
of mVp8.2b to Mls-la. Additional studies are required
to accurately assign the V, binding sites for MAM and
other superantigens.
Recent reports indicate that N-glycosylation
sites may also affect binding of superantigens to &
(38). However, since the MAM-engaged mV,8.3 and
the MAM-nonengaged rV,8.3 had identical glycosylation sites, differences in MAM reactivities between
these 2 V, cannot be attributed to steric hindrance
conferred by N-linked carbohydrates.
Initial experiments with I-E- mice of the H-2b
haplotype (EKE@+)(9) and more recent studies with
transfected fibroblasts and E a transgenic H-2b mice
(39), indicated that the MHC binding site for MAM
resides in the I-E a chain. Our present study with
splenocytes of Ea-EP- mice transgenic for human
DR molecules indicate that MAM presentation is also
primarily determined by the DR a chain, the human
homolog of mouse Ea. Based on the structural model
described by Brown et a1 (44) for class I1 MHC
molecules, Ea and DRa exhibit identity at a continuous stretch of 20 amino acids constituting most of the
putative a helix, which through different contact
points (17), might be involved in the binding of both
conventional peptides and superantigens. Although
this and other studies indicate that DRa or I-Ea in
humans and mice, respectively, are the principal molecules via which MAM binds and is presented to T
cells, it cannot be excluded that some T cell engagements might occur via MAM binding to other cell
surface molecules.
Remarkably, the set of mouse V, engaged when
MAM was presented in the context of mouse E a is
identical to that when MAM is presented in the context of human DRa. This finding is consistent with the
439
studies by Lawrence et al(24), who found that expression of human DRa in Ea- EP+ transgenic mice led to
Vp clonal deletions identical to those mediated by
endogenous superantigens in I-E+ mice. These findings, however, contrast somewhat with those of Herman et a1 (49, who reported that presentation of
staphylococcal enterotoxins in the context of human
DR resulted in a broader mV, engagement and included mV, that were not engaged when presentation
was in the context of mouse class I1 MHC molecules.
It was suggested that such broader reactivities may be
caused by the higher affinity of these human pathogens
(staphylococcal toxins) for human, rather than mouse,
MHC molecules, leading to increased ligand concentrations and subsequent T cell responses. Obviously,
this does not appear to be the case for MAM (the
product of a mouse pathogen; present study) or
MMTV-related superantigens (ref. 24 and unpublished
data). We also found that transgenic splenocytes expressing the nonpolymorphic DRa associated with
either of the highly polymorphic A P or DRP, were
equally effective in eliciting strong V,-specific responses to MAM. These results suggest that responses
to MAM in humans, unlike the responses to staphylococcal enterotoxins that are influenced by the polymorphic DRP (49, may be DR haplotype-independent.
MAM is known to contribute to lethal toxicity
and dermal necrosis induced by M arthritidis in mice
(23) and will induce transient arthritis when given
intraarticularly (46). However, there is presently no
formal proof that superantigens are involved in the
pathogenesis of chronic arthritis in animals or humans.
Nevertheless, several mechanisms may be envisaged
by which such molecules could cause joint inflammation. They include cell-mediated mechanisms, such as
release of large amounts of cytokines (47), induction of
cytotoxic responses against self-class I1 MHCexpressing cells (48-50), and cross-reactivity of activated T cells with synoviocytes (51), and/or humoral
mechanisms, such as polyclonal autoantibody induction by superantigen-presenting B cells collaborating
with the Vp-engaged T cells (52).
Based on these considerations, attempts have
been made to characterize the T cell clonotypes associated with synovial infiltrates in patients with RA.
Although the findings are as yet inconclusive, evidence has been gathered for the involvement of oligoclonal sets of T cells (53-55). It is worth noting that in
one of these studies ( 5 9 , the predominant T cell
clonotypes identified in the synovium of a few RA
patients expressed vB3.1 and V,19.l (or, V,17.1), the
BACCALA ET AL
440
exact V, shown in this study to be MAM-responsive.
However, the V, identified differ considerably from
one study to another, clonotypes appear to be unique
in each patient, and repeated usage was applicable not
only to the V,, but also to specific D, and J, (5435).
This latter finding is evidence against the involvement
of superantigens, since the major characteristic of
these molecules is the engagement of specific V,,,
regardless of the associated DBJp elements (19). Alternatively, however, as previously suggested (541,
encounter of such a superantigen in the circulation
may be the initiating event that subsequently leads to
the sequestration in the joint of a few activated cells
that display restricted VrDrJp sets of receptors with
affinity for a cross-reacting synovium-associated conventional peptide antigen. Analysis of synovial T cell
clonotypes of a larger population of RA patients
together with appropriate studies in animal models of
Mycoplusma-induced arthritis and other superantigeninduced arthritis will be required to clarify whether
superantigens and the attendant V, engagements are
indeed involved in arthritis induction.
Addendum. While this paper was under review,
Friedman et a1 (561, using a monoclonal antibody, also
reported engagement of human V,19.1 (alternatively, V,17. I )
by MAM.
ACKNOWLEDGMENTS
We thank Drs. D. Kono, M. Kammuller, and R. S.
Balderas for assistance with various aspects of this work.
and M. K. Occhipinti for editing and manuscript production.
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